CN112553121A - Ultrahigh-density solid fermentation method for high-temperature-resistant bacillus - Google Patents
Ultrahigh-density solid fermentation method for high-temperature-resistant bacillus Download PDFInfo
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- CN112553121A CN112553121A CN202011560636.3A CN202011560636A CN112553121A CN 112553121 A CN112553121 A CN 112553121A CN 202011560636 A CN202011560636 A CN 202011560636A CN 112553121 A CN112553121 A CN 112553121A
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- 238000000855 fermentation Methods 0.000 title claims abstract description 58
- 230000004151 fermentation Effects 0.000 title claims abstract description 58
- 239000007787 solid Substances 0.000 title claims abstract description 52
- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 title claims abstract description 17
- 239000001963 growth medium Substances 0.000 claims abstract description 27
- 239000007788 liquid Substances 0.000 claims abstract description 24
- 238000012258 culturing Methods 0.000 claims abstract description 17
- 229920001817 Agar Polymers 0.000 claims abstract description 14
- 239000008272 agar Substances 0.000 claims abstract description 14
- 230000003213 activating effect Effects 0.000 claims abstract description 5
- 239000002609 medium Substances 0.000 claims description 16
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 6
- 239000002994 raw material Substances 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 235000008733 Citrus aurantifolia Nutrition 0.000 claims description 4
- 235000019764 Soybean Meal Nutrition 0.000 claims description 4
- 235000011941 Tilia x europaea Nutrition 0.000 claims description 4
- 240000008042 Zea mays Species 0.000 claims description 4
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 4
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 4
- 235000005822 corn Nutrition 0.000 claims description 4
- 235000013312 flour Nutrition 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 239000004571 lime Substances 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 239000004455 soybean meal Substances 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 4
- 239000002068 microbial inoculum Substances 0.000 abstract description 5
- 239000003795 chemical substances by application Substances 0.000 abstract description 4
- 239000010806 kitchen waste Substances 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 239000006137 Luria-Bertani broth Substances 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- 125000004122 cyclic group Chemical group 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000012271 agricultural production Methods 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000002361 compost Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 210000003278 egg shell Anatomy 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 235000021190 leftovers Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000010813 municipal solid waste Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000003895 organic fertilizer Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Processing Of Solid Wastes (AREA)
Abstract
The invention discloses a high-temperature-resistant bacillus ultrahigh-density solid fermentation method, which comprises the following steps: (1) activating strains: culturing high-temperature resistant bacillus in an LB solid culture medium to obtain an activated strain; (2) preparing liquid seed liquid: inoculating the activated strain obtained in the step (1) into a liquid LB culture medium for culture; (3) solid fermentation: and (3) inoculating the liquid seed solution prepared in the step (2) into a solid fermentation culture medium, and fermenting to obtain the high-density bacillus, wherein the fermentation culture medium comprises agar. According to the invention, agar is added into the optimized solid culture medium as a water-retaining agent, so that the ultrahigh-density solid fermentation of the high-temperature-resistant bacillus is realized, and the colony number of the final microbial inoculum product greatly exceeds the standard of the microbial inoculum and can reach 100 hundred million CFU/g.
Description
Technical Field
The invention relates to the technical field of kitchen waste treatment, in particular to a high-temperature-resistant bacillus ultrahigh-density solid fermentation method.
Background
The kitchen waste mainly refers to kitchen waste generated in activities such as daily life, food processing, food service, unit catering and the like of residents, and comprises discarded leftovers, fruit peels, eggshells, bones and the like. Traditional modes such as landfill, burning and feeding pig can't satisfy people to health, and resource cyclic utilization's requirement, so can adopt kitchen garbage treatment equipment to carry out dehydration at present, and organic fertilizer is produced to rethread biological fermentation, carries out agricultural production, but realizes the cyclic utilization of resource. However, considering the characteristics of high oil content, high salt content and the like of the kitchen waste, which can cause poor compost quality, high-temperature bacteria capable of resisting salt and reducing oil are needed for biological fermentation, the high-temperature bacteria not only have strong metabolic capability in a high-temperature environment, but also greatly shorten the biological conversion time of the kitchen waste, and improve the degradation efficiency of organic matters.
At present, the fermentation modes of the microbial inoculum mainly comprise liquid fermentation and solid fermentation, the liquid fermentation needs to be dried after fermentation, the energy consumption is large, the production cost is improved, the process procedures are more, and the requirements on personnel and equipment are higher; the solid fermentation does not need to consume huge time and electric quantity for drying after fermentation, can be directly dried and crushed for use, has relatively simple process, less labor and equipment cost, and greatly saves the investment cost.
However, for the fermentation of high temperature resistant microorganisms, the water content of the fermentation medium is also met while the high temperature condition is maintained, so that the microorganisms have relatively sufficient activity space to utilize the nutrient substances in the medium. Therefore, the water-retaining agent is added into the solid fermentation culture medium to maintain the good environment for the living of the microorganisms.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the technical problem of providing a high-temperature-resistant bacillus ultrahigh-density solid fermentation method.
The technical scheme is as follows: in order to solve the technical problems, the invention adopts the following technical scheme:
a high-temperature-resistant bacillus ultrahigh-density solid fermentation method comprises the following steps:
(1) activating strains: culturing high-temperature resistant bacillus in an LB solid culture medium to obtain an activated strain;
(2) preparing liquid seed liquid: inoculating the activated strain obtained in the step (1) into a liquid LB culture medium for culture;
(3) solid fermentation: inoculating the liquid seed solution prepared in the step (2) into a solid fermentation culture medium, and fermenting to obtain high-density bacillus;
the preparation method of the solid fermentation medium comprises the following steps: weighing the following materials in parts by weight: 60-70 parts of bran, 20-25 parts of soybean meal, 5-10 parts of corn flour and 3-5 parts of calcium carbonate, uniformly mixing all the raw materials in a stirrer, adding water according to the proportion of 1g to 1.5 mL, then adding 5g/L agar powder, adjusting the pH value to 7 with lime, and sterilizing to obtain the solid fermentation medium.
In the step (1), the time for culturing the high-temperature resistant bacillus in the LB solid culture medium is 20-25 h.
In the step (2), the conditions for culturing in the LB medium are as follows: culturing at 37-60 ℃ for 3-5 days at a speed of 150-200 r/min.
In the step (3), the inoculation amount of the seed liquid is 5-7% of the mass of the solid fermentation medium.
In the step (3), the fermentation conditions of the fermentation are as follows: culturing at 50-60 ℃ for 3d at 200r/min and 180-.
Has the advantages that:
according to the invention, the formula of the solid culture medium is optimized, the ultrahigh-density solid fermentation of the high-temperature-resistant bacillus is realized, the colony number of the final microbial inoculum product greatly exceeds the standard of the microbial inoculum and can reach 100 hundred million CFU/g, the water-retaining agent agar is added into the culture medium, the excessive evaporation of water in the high-temperature fermentation process is prevented, the energy and the cost of continuously adding water in the middle of manual work are reduced, the ultrahigh-density raw material is low in price, the operation is simple and convenient, no danger is generated, and the method is suitable for large-scale production. The agar is used as a common reagent, is nontoxic and harmless to the growth of microorganisms, is cheap and easy to obtain, does not pollute the environment, and can be used as a water-retaining agent for ultrahigh-density solid fermentation.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Example 1:
1. activating strains: LB broth 20g/L, agar 20g/L, pH 7, LB solid medium was prepared and sterilized. Taking out the strain preservation tube preserved in a refrigerator at the temperature of 20 ℃ below zero in advance until the strain is frozen and completely dissolved. Inoculating the strain to LB solid culture medium by plate-drawing method in sterile super clean bench, and culturing at 60 deg.C for 20 hr to obtain activated strain.
2. Ultra high density fermentation
(1) Preparation of liquid seed liquid
3g/L of beef extract, 10g/L of peptone, 5g/L of NaCl, and the pH =7, and sterilizing for later use. Inoculating the activated strain into 50mL of liquid culture medium, and culturing in a shaker at 60 ℃ and 180r/min for 3d for later use.
(2) Preparation of solid fermentation Medium
Weighing the following materials in percentage by weight: 60 parts of bran, 25 parts of soybean meal, 10 parts of corn flour and 5 parts of calcium carbonate, putting all the raw materials into a dry stirrer, fully and uniformly mixing the raw materials at the rotating speed of 30r/min, positively rotating for 10min and reversely rotating for 10min, adding a certain amount of water according to the proportion of 1:1.5 (g/mL), then adding a certain amount of agar powder with the concentration of 5g/L, and adding a certain amount of lime to adjust the pH value to 7. Sterilizing the solid fermentation culture medium in autoclave for 20min with parameters of 121 deg.C and 1.2kg/cm3。
(3) Solid fermentation
Inoculating the seed liquid prepared in the step (1) into the solid fermentation culture medium in the step (2), wherein the inoculation amount of the seed liquid is 7% of the mass of the solid fermentation culture medium, fully and uniformly stirring, putting the solid fermentation culture medium into a shaking table at 180r/min and 60 ℃ for culturing for 3d, naturally airing at normal temperature, and determining the colony number to be 128 hundred million CFU/g.
Example 2:
1. activating strains: LB broth 20g/L, agar 25g/L, pH 7, LB solid medium was prepared and sterilized. Taking out the strain (ORI-1) in the refrigerator at-80 deg.C in advance, and freezing to dissolve completely. Inoculating the strain to LB solid culture medium by plate-drawing method in sterile super clean bench, and culturing at 50 deg.C for 24 hr to obtain activated strain.
2. Ultra high density fermentation
(1) Preparation of liquid seed liquid
3g/L of beef extract, 10g/L of peptone, 5g/L of NaCl, and =7 of ph, and sterilizing for later use. Inoculating the activated strain into 100mL of liquid culture medium, and culturing for 5d at 50 ℃ in a shaking table at 180r/min for later use.
(2) Preparation of solid Medium
Weighing the following materials in percentage by weight: bran 65%, soybean meal 25%, corn flour 7% and calcium carbonate 3%, putting all the raw materials into a dry stirrer, fully and uniformly mixing at a rotation speed of 30r/min, positively rotating for 10min and reversely rotating for 10min, adding a certain amount of water according to a ratio of 1:2 (g/mL), then adding a certain amount of agar powder, wherein the concentration of agar is 10g/L, and adding a certain amount of lime to adjust the pH value to 8. Placing the solid fermentation medium in an autoclave for sterilization for 30min, setting the parameters at 121 deg.C and 1.0kg/cm 3.
(3) Solid fermentation
Inoculating the seed liquid prepared in the step (1) into the solid fermentation culture medium in the step (2), wherein the inoculation amount of the seed liquid is 5% of the mass of the solid fermentation culture medium, fully and uniformly stirring, putting the solid fermentation culture medium into a shaking table at 180r/min and 50 ℃ for culturing for 5 days, naturally airing at normal temperature, and determining the colony number to be 115 hundred million CFU/g.
Comparative example 1: the experiment was carried out according to the protocol of example 2, without agar addition, the final number of colonies was 3 hundred million CFU/g.
Comparative example 2: the experiment was carried out according to the protocol of example 2, without agar addition, the final number of colonies was 2 hundred million CFU/g.
As can be seen from the results of examples 1-2 and comparative examples 1-2, the water retention capacity of the culture medium can be effectively improved and the fermentation density can be improved after the agar is added.
Claims (5)
1. The ultrahigh-density solid fermentation method of the high-temperature-resistant bacillus is characterized by comprising the following steps of:
(1) activating strains: culturing high-temperature resistant bacillus in an LB solid culture medium to obtain an activated strain;
(2) preparing liquid seed liquid: inoculating the activated strain obtained in the step (1) into a liquid LB culture medium for culture;
(3) solid fermentation: inoculating the liquid seed solution prepared in the step (2) into a solid fermentation culture medium, and fermenting to obtain high-density bacillus;
the preparation method of the solid fermentation medium comprises the following steps: weighing the following materials in parts by weight: 60-70 parts of bran, 20-25 parts of soybean meal, 5-10 parts of corn flour and 3-5 parts of calcium carbonate, uniformly mixing all the raw materials in a stirrer, adding water according to the proportion of 1g to 1.5 mL, then adding 5g/L agar powder, adjusting the pH value to 7 with lime, and sterilizing to obtain the solid fermentation medium.
2. The ultrahigh-density solid fermentation method of high temperature resistant bacillus of claim 1, wherein in the step (1), the time for culturing the high temperature resistant bacillus in the LB solid medium is 20-25 h.
3. The ultrahigh-density solid fermentation method of high temperature resistant bacillus according to claim 1, wherein in the step (2), the conditions for culturing in LB medium are: culturing at 37-60 ℃ for 3-5 days at a speed of 150-200 r/min.
4. The ultrahigh-density solid fermentation method of high-temperature-resistant bacillus according to claim 1, wherein in the step (3), the inoculation amount of the seed liquid is 5-7% of the mass of the solid fermentation medium.
5. The ultrahigh-density solid fermentation method of high temperature resistant bacillus according to claim 1, wherein in the step (3), the fermentation conditions of the fermentation are as follows: culturing at 50-60 ℃ for 3d at 200r/min and 180-.
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|---|---|---|---|---|
| CN103205388A (en) * | 2013-05-08 | 2013-07-17 | 东莞市保得生物工程有限公司 | Method for producing viable bacteria of bacillus licheniformis by high-density solid fermentation |
| CN103289937A (en) * | 2013-06-18 | 2013-09-11 | 东莞市保得生物工程有限公司 | Method for producing bacillus laterosporus live bacteria by high density solid fermentation |
| CN103468613A (en) * | 2013-09-12 | 2013-12-25 | 江苏省苏微微生物研究有限公司 | Bacillus megatherium, method for preparing microbial inoculum through solid fermentation of bacillus megatherium and application of microbial inoculum |
| CN104388347A (en) * | 2014-11-18 | 2015-03-04 | 安佑生物科技集团股份有限公司 | Solid-state fermentation method for feeding bacillus subtilis with high germination rate and high stress resistance |
| CN110964677A (en) * | 2020-01-20 | 2020-04-07 | 江苏兴鼎生物工程有限公司 | Method for solid fermentation of bacillus coagulans |
-
2020
- 2020-12-25 CN CN202011560636.3A patent/CN112553121A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103205388A (en) * | 2013-05-08 | 2013-07-17 | 东莞市保得生物工程有限公司 | Method for producing viable bacteria of bacillus licheniformis by high-density solid fermentation |
| CN103289937A (en) * | 2013-06-18 | 2013-09-11 | 东莞市保得生物工程有限公司 | Method for producing bacillus laterosporus live bacteria by high density solid fermentation |
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| CN110964677A (en) * | 2020-01-20 | 2020-04-07 | 江苏兴鼎生物工程有限公司 | Method for solid fermentation of bacillus coagulans |
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| HARMEET K. SODHI等: "Production of a thermostable -amylase from Bacillus sp. PS-7 by solid state fermentation and its synergistic use in the hydrolysis of malt starch for alcohol production", 《PROCESS BIOCHEMISTRY》 * |
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Application publication date: 20210326 |